Influence of different noxa on DNA repair capacity of primary human skin cells

The human genome provides the basis for reproduction, proper development and correct functioning of the whole organism. Despite this, DNA is constantly under attack of damaging chemical or physical influences. These extrinsic influences as well as intrinsic processes give rise to DNA damages possibly ending in mutations during replication.

In the course of evolution several highly specialized repair pathways have evolved to counteract DNA damages and therefore preserve the integrity of the genome. The importance of repair pathways is impressively illustrated by rare repair disorders like Xeroderma Pigmentosum. Affected individuals possess severely limited nucleotide excision repair and therefore show a 1000-fold increased cancer risk. For this reason, DNA repair pathways are promising targets with regard to the development of protective agents as well as with regard to the identification of potentially harmful substances.

The importance of influences on repair pathways is reflected by an OECD test guideline, published in 1997 describing the UDS repair assay in the context of DNA damages and repair to determine genotoxic properties.

Aim of this study was to establish a considerably more sensitive, reproducible, non-radioactive assay to determine influences on NER capacity. This assay should neither afford an inhibition of replication nor exogenous impairment of the cell population. It should be suitable for studying biocompatibility/genotoxicity as well as for screening for possible active ingredients.

Within the framework of cooperation with an industrial company we established a host cell reactivation assay to determine the repair capacity of primary skin cells meeting the claims made. The utilization of different primary skin cells derived from a single skin biopsy poses a challenge particularly with regard to isolation, maintenance and transfection procedures. For the first time fluorescence activated cell sorting was used for repair capacity analysis providing a single cell based interpretation of large cell populations which is reflected in an increased sensitivity. This method was published under the title >>A modified host cell reactivation assay to determine the repair capacity of primary human keratinocytes, melanocytes and fibroblasts<< (Burger et al.).Using this method, we showed a negative influence of Cyclosporin A on the NER of skin cells, providing a clue for the involvement of NER in enhanced cancer risk following Cyclosporin A therapy. Several other studies corroborate this believe.

In addition we demonstrated a negative influence of folic acid depletion on the repair capacity skin cells in a study involving 17 donors. Each individual showed a considerable decline of repair capacity following folic acid depletion. The results of this study were published under the title >>The influence of folic acid depletion on the nucleotide excision repair capacity of human dermal fibroblasts measured by a modified host cell reactivation assay<< (Burger et al.)

One further study concerned the influences of low dose UV radiation on the DNA repair capacity. We detected a significant increase of NER capacity following preirradiation of the cell populations with very low UV doses. This effect couldn’t be boosted by further low dose irradiations at intervals of 24 h. In the context of this study, we additionally analyzed alterations of the gene expression pattern of different NER genes following low dose UV radiation. The results of this study were summarized to a manuscript titled >>Low doses of UVB enhance reactivation of UV damaged reporter gene in dermal fibroblasts<< (Burger et al.).

Taken together the new assay system to detect influences on DNA repair capacity implies a significant progress in the context of design and development of protective agents (screening for active ingredients) as well as in context of identifying negative influences on DNA repair capacity (detection of genotoxicity). This system provides potential for development as to higher throughput of potential agents and to influences on other repair pathways than nucleotide excision repair.

2011-08-31T09:54:43ZBurger, KatharinaInfluence of different noxa on DNA repair capacity of primary human skin cellsBurger, Katharinaterms-of-use2011eng2011-08-31T09:54:43ZThe human genome provides the basis for reproduction, proper development and correct functioning of the whole organism. Despite this, DNA is constantly under attack of damaging chemical or physical influences. These extrinsic influences as well as intrinsic processes give rise to DNA damages possibly ending in mutations during replication.<br /><br />In the course of evolution several highly specialized repair pathways have evolved to counteract DNA damages and therefore preserve the integrity of the genome. The importance of repair pathways is impressively illustrated by rare repair disorders like Xeroderma Pigmentosum. Affected individuals possess severely limited nucleotide excision repair and therefore show a 1000-fold increased cancer risk. For this reason, DNA repair pathways are promising targets with regard to the development of protective agents as well as with regard to the identification of potentially harmful substances.<br /><br />The importance of influences on repair pathways is reflected by an OECD test guideline, published in 1997 describing the UDS repair assay in the context of DNA damages and repair to determine genotoxic properties.<br /><br />Aim of this study was to establish a considerably more sensitive, reproducible, non-radioactive assay to determine influences on NER capacity. This assay should neither afford an inhibition of replication nor exogenous impairment of the cell population. It should be suitable for studying biocompatibility/genotoxicity as well as for screening for possible active ingredients.<br /><br />Within the framework of cooperation with an industrial company we established a host cell reactivation assay to determine the repair capacity of primary skin cells meeting the claims made. The utilization of different primary skin cells derived from a single skin biopsy poses a challenge particularly with regard to isolation, maintenance and transfection procedures. For the first time fluorescence activated cell sorting was used for repair capacity analysis providing a single cell based interpretation of large cell populations which is reflected in an increased sensitivity. This method was published under the title >>A modified host cell reactivation assay to determine the repair capacity of primary human keratinocytes, melanocytes and fibroblasts<< (Burger et al.).Using this method, we showed a negative influence of Cyclosporin A on the NER of skin cells, providing a clue for the involvement of NER in enhanced cancer risk following Cyclosporin A therapy. Several other studies corroborate this believe.<br /><br />In addition we demonstrated a negative influence of folic acid depletion on the repair capacity skin cells in a study involving 17 donors. Each individual showed a considerable decline of repair capacity following folic acid depletion. The results of this study were published under the title >>The influence of folic acid depletion on the nucleotide excision repair capacity of human dermal fibroblasts measured by a modified host cell reactivation assay<< (Burger et al.)<br /><br />One further study concerned the influences of low dose UV radiation on the DNA repair capacity. We detected a significant increase of NER capacity following preirradiation of the cell populations with very low UV doses. This effect couldn’t be boosted by further low dose irradiations at intervals of 24 h. In the context of this study, we additionally analyzed alterations of the gene expression pattern of different NER genes following low dose UV radiation. The results of this study were summarized to a manuscript titled >>Low doses of UVB enhance reactivation of UV damaged reporter gene in dermal fibroblasts<< (Burger et al.).<br /><br />Taken together the new assay system to detect influences on DNA repair capacity implies a significant progress in the context of design and development of protective agents (screening for active ingredients) as well as in context of identifying negative influences on DNA repair capacity (detection of genotoxicity). This system provides potential for development as to higher throughput of potential agents and to influences on other repair pathways than nucleotide excision repair.